Yeast fatty acid synthetase

Sedgwick B, Morris C, French SJ. Stereochemical course of 45. dehydration catalyzed by yeast fatty-acid synthetase. J. Chem. Soc. 

It has been reported that, like liver acetyl-CoA carboxylase, both the liver and yeast fatty acid synthetases are inhibited by low concentrations (0.5 to 5 X 10 71/) of long-chain fatty acyl-CoA derivatives, the longer-chain derivative producing greater inhibition [226,246,247]. In the case of the yeast synthetase, inhibition by long-chain acyl-CoA derivatives was competitive with respect to acetyl-CoA and NADPH. For the same reasons alluded to earlier in the discussion of the inhibition of acetyl-CoA carboxylase by fatty acyl-CoA derivatives, some caution must be exercised in interpreting the effect of these potent inhibitors (see Section V, C, 2). 

G.B. Kresze, L. Steber, E. Lynen, D. Oesterhelt, Reaction of yeast fatty-acid synthetase with iodoacetamide. 3. Malonyl-coenzyme-a decarboxylase as product of reaction of fatty-acid synthetase with iodoacetanride. Eur. J. Biochem. 79, 191-199 (1977)... 

Analogy between 6-Methylsalicylic Acid Synthetase and Yeast Fatty Acid Synthetase... 

Fatty acid synthetases are divided into Type I, Type II and Type III. Type I enzymes are multifunctional proteins which contain covalently bound acyl carrier protein (ACP) and release products as unesterified fatty acids (mammals) or transfer them to Co A (yeast). In animals the fatty acid synthetase is a homo dimer with each chain containing all of the partial reactions. By contrast, yeast fatty acid synthetase contains two dissimilar peptides and operates as an complex. 

In the organism tissues, fatty acids are continually renewed in order to provide not only for the energy requirements, but also for the synthesis of multicomponent lipids (triacylglycerides, phospholipids, etc.). In the organism cells, fatty acids are resynthetized from simpler compounds through the aid of a supramolecular multienzyme complex referred to as fatty acid synthetase. At the Lynen laboratory, this synthetase was first isolated from yeast and then from the liver of birds and mammals. Since in mammals palmitic acid in this process is a major product, this multienzyme complex is also called palmitate synthetase. 

Figure 11.2 A comparative picture of the fatty acid synthetase (FAS) systems in yeast, animal, bacterial and plant cells. fi-KS, -ketoacyl AGP synthetase P-KR, A-ketoacyl AGP reductase DH, -OH acyl-AGP dehydrase ER, enoyl AGP reductase AT, acetyl transacylase MT, malonyl transacylase TE, thioesterase AGP, acyl carrier protein. See Shimakata and Stumpf (19S2a,b) and Wakil etal, (1983) for details.

Partial reactions for the yeast Type I synthetase were studied in a classic series of experiments by Lynen (1967). Peptides capable of partial reactions have been isolated from the yeast and chicken liver enzymes (cf. Wakil et ai, 1983). Particular interest has been focused on the thioesterase. This enzyme, more easily isolated by partial proteolysis than those for most of the other partial reactions, is important in at least partly regulating chain length termination. Thioesterases isolated from several animal fatty acid synthetases have very similar amino acid sequences around the active serine residue (Poulose et al, 1981). The medium-chain fatty acids produced by synthetases from some mammary glands appear to be due to thioesterase II (a second thioesterase) (Libertini and Smith, 1978). 

Fatty acids in plants are synthesized by a series of reactions similar to those for fatty acid biosynthesis in animals, yeasts, bacteria, and other organisms. The synthetases of prokaryotic cytoplasmic systems are freely soluble and readily separable as discrete proteins those in mammalian systems are localized in the cytoplasm as large soluble synthetase complexes. The exact nature of association of the enzymes in plants is unknown (Ohlrogge et al., 1993). The reactions of plant fatty acid biosynthesis are catalyzed by individual polypeptides (Somerville and Browse, 1991) once organelles are disrupted, the enzymes also are soluble and may be isolated (Somerville and Browse, 1991 Stumpf, 1976). In contrast to mammalian systems, plant fatty acid synthetases are plastid localized. 

In prokaryotic cells fatty acid synthesis occurs in the cytosolic compartment. However, it has been observed that ACP in E. coli appears to be somewhat loosely associated with the inner face of the plasma membrane of the cell (van den Bosch et al., 1970). Nevertheless, all the activities associated with the synthesis of palmitic acid from acetyl-CoA can be readily separated and assigned to individual proteins which have been purified and their molecular and kinetic characteristics examined in considerable detail (Vagelos, 1974). In yeast and animal cells, the fatty acid synthetase responsible for the formation of palmitic acid is always associated with the cytosolic compartment as a dimer of a polyfunctional polypeptide (ibid.). 

Since fatty acid and aromatic synthetases have many properties in common, it is interesting to consider the effect of omitting NADPH from incubations catalyzed by these enzymes and to examine the consequences. In all cases studied from animal (Bressler and Wakil, 1%2 Nixon et al., 1%8), yeast (Yalpani et al., 1969), and bacterial origin (Brock and Bloch, 1966), fatty acid synthesis is naturally abolished, but a Q compound, triacetic acid lactone (4-hydroxy-6-methyl-2-pyrone) (VII), is formed instead. This product is also made when the NADPH-requiring 6-methylsalicylate synthetase is deprived of this nucleotide (Dimroth et al., 1972), but the relative rate of synthesis is considerably greater than that produced by fatty acid synthetase in P. patulum (Yalpani et al., 1%9). However, Scott et al. (1971) have reported that triacetic acid lactone is also formed by 6-methylsalicylate synthetase, albeit at a reduced rate, even in the presence of NADPH. 

Fatty Acid Synthetase in Yeast and Avian and Mammalian Tissues... 

This is distinct from the type 1, associated fatty acid synthetase, which is exemplified by both yeast - in which the activities are present on two polypeptide chains and animals - in which all the activities are present on one polypeptide. Understanding the nature of these enzymes and the way in which they function has moved a long way from the early days of enzyme isolation and cDNA cloning. With the ability to overproduce proteins in bacterial (Kater. M. M et al., 1991) yeast and baculovirus systems (Joshi.A. K Smith. S., 1993)limitations on the availability of enzyme for X-ray crystallographic studies are largely overcome. Providing the protein will cryst lise then a solution to the structure is possible. 

The enzyme system which converts malonyl-CoA to higher fatty acids, referred to as fatty acid synthetase , behaved as a single entity during the fractionation of tissue extracts and was isolated in a number of laboratories from various animal tissues. We ourselves obtained highly active enzyme fractions from yeast. ... 

The two ketoacyl-synthetases present in the fatty-acid synthetase complexes of higher plants exhibit differential sensitivities against cerulenin. In plants the B-ketoacyl-synthetase I (de novo fatty acid synthesis) is affected by cerulenin, whereas the B-ketoacylsynthetase II, which catalyzes the elongation from 16 0 to 18 0 is relatively insensitive (Jaworski et al. 1974). The B-ketoacylsynthetase of Cephalosporium caerulens, which produces cerulenin, is much less sensitive than the yeast enzyme (Kawaguchi et al. 1979) and in Escherichia coli two sensitive and one insensitive synthetase were described (Jackowski and Rock 1987). 

F. Lynen, H. Engeser, J. Friedrich, W. Schindlbeck, R. Seyffert, and F. Wieland, Fatty acid synthetase of yeast and 6-methylsalicylate synthetase of Penicillium patulum - two multienzyme complexes, iji "Microenvironments and Metabolic Conpartmentation," P.A. Srere and R.W. Estabrook, eds.. Academic Press, New York (1978). 

The 4 -phosphopantotheine residue has been identified as part of the enzyme (Dimroth et aL, 1972). This identifies the same acyl carrier flexible arm which has been isolated in fatty acid synthetase from yeast and Escherichia coli. 

Six separate enzyme activities are involved and the steps have been elucidated mainly from studies of E. coli, yeast, the tissues of various animals and some higher plants. The group of enzymes are known collectively as fatty acid synthetase and it is now clear that, although the overall biochemistry is the same in all cases, the organization of the synthetases is very different. 

Fatty acid synthetases can be divided mainly into Type I and Type II enzymes (Table 3.8). Type I synthetases are multifunctional proteins in which the proteins catalysing the individual partial reactions are discrete domains. This type includes the animal synthetases and those from higher bacteria and yeast. Type II synthetases contain enzymes which can be separated, purified and studied individually. This system occurs in lower bacteria and plants and has been studied most extensively in E. coli. In addition, Type III synthetases - occurring in different organisms - catalyse the addition of C2 units to preformed acyl chains and are also known as elongases. Although, historically, the reactions of the yeast synthetase were unravelled first, we shall start by describing the separate reactions catalysed by the enzymes of E. coli. 

Figure 3.7 Model of intermolecular fatty acid synthetase mechanism in the a2 2 protomer of yeast. A, acetyl transferase E, enoyl reductase D, dehydratase P, palmitoyl transferase M, malonyl transferase C, 5-ketoacyl synthase R. )5-ketoacyl reductase ACP, acyl carrier protein. Dotted lines and arrows delineate the route taken by intermediates when sequentially processed on different FAS domains. Numbers indicate the reaction sequence. Catalytically active dohnains, at a specific moment, are marked by bold lines. Shaded areas on E and P domains potentially interact by hydrophobic attraction in the presence of palmitate (b). On the protomer depicted in (a) fatty acyl chain elongation occurs in one half of the a2 2 protomer. In (b) chain termination is induced by hydrophobic interaction between E> bound palmitate and P. Subsequently, palmitate Is transferred to Its O-ester binding site on P. Inactivation of the left half of simultaneously activates its right half (b). Redrawn from Schweizer (1984) with permission of the author and Elsevier Science Publishers, BV. From Fatty Acid Metabolism and its Regulation (1984) (ed. S. Numa), p. 73, Figure 7.

Basically, there are two completely different pathways by which unsaturated fatty acids are produced. In an earlier section, we mentioned that the fatty acid synthetase of E, coli, in contrast to the mammalian and yeast synthetases, produced unsaturated as well as saturated acids. An idea of... 

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